Similar to the mammalian hippocampus, Drosophila mushroom bodies (MBs) are critical for learning and memory. Surprisingly, the neurotransmitter(s) stored and released by the intrinsic neurons of the MBs, the Kenyon cells (KCs) are not known. We recently identified a novel vesicular neurotransmitter transporter, portabella (prt) that is expressed in KCs. We propose to use prt as a tool to identity the neurotransmitter stored in KCs. We will use two complementary methods. In Aim 1, we will perform in vitro transport assays using cells that express prt. In Aim 1A, we will test transport o radiolabeled candidates. Aim 1B describes a backup plan using a technique that does not require radiolabeled compounds. Since 1B is more risky than 1A it will only be employed if both Aim 1A and Aim 2 fail. In Aim 2 we will assay the lumenal contents of secretory vesicles using electrochemical detection and mass spectrometry. In Aim 2A we will biochemically fractionate secretory vesicles. We hypothesize that the small molecule stored by prt in wt flies will be reduced in vesicles derived from the prt mutant. As a backup plan (Aim 2B), we will assay the exocytotically released contents of secretory vesicles from cultured KCs. We hypothesize that a small molecule released into the media by wt KCs will not be released by prt mutant neurons. Candidate molecules identified in Aim 2 (or 1B) will be directly tested in transport assays as in Aim 1A. The results of these experiments will determine the prt substrate. Since prt is the only vesicular neurotransmitter that is expressed in KCs, these experiments will identify the neurotransmitter(s) that is stored in KCs.